Ecdysteroid kinase in silkworm eggs : Purification and characterization

蚕卵中的蜕皮类固醇激酶：纯化和表征

• 批准号: 13640672
• 负责人: SONOBE Haruyuki
• 金额: $ 2.24万
• 依托单位: Konan University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13640672/
• 关键词: ecdysteroids; Ecdysteroid-phosphate; kinase; silkworm; カイコ; エクジステロイド受容体

In eggs of many insect species, ecdysteroid-phosphates, which are of maternal origin, have widely been accepted as storage forms for supplying active free ecdysteroids during embryonic development (Makka et al., 2002). However, there is little information on the enzyme which specifically catalyzes the phosphorylation of free ecdysteroids. The purpose of this study is to purify and characterize the ecdysteroid kinase (ATP : ecdysteroid phosphotransferase).Ecdysteroid kinase was assayed by the phosphorylation reaction of [^3H] ecdysone. The enzyme, which was located in the cytosol fraction in mature ovaries of the silkworm Bombyx mori, was purified by about 600-fold to homogeneity by seven steps of column chromatography : Q Sepharose, Chelating Sepharose, Hydroxylapatite, Hitrap Q, Mono Q, 5'AMP Sepharose 4B and Superdex 200pg. The purified enzyme was most active at pH 7.6. The enzyme required the presence of both ATP and Mg^<2+> for expressing the activity. The apparent kinetic parameters for ecdysone kinase was Km=3.30 μM (20-hydroxyecdysone), 3.66 μM (ecdysone), 8.22 μM (3-epiecdysone). It is not clear at present whether ATP : 2-deoxyecdysone 22-phosphotransferase found in Schistocerca gregaria (Kabbouh and Rees 1991) and our ecdysone 22-phosphotransferase (ecdysone kinase) in Bombyx mori are homologous.

在许多昆虫物种的卵中，母体来源的蜕皮类固醇-磷酸盐已被广泛接受为用于在胚胎发育期间提供活性游离蜕皮类固醇的储存形式（Makka等人，2002年）。然而，有很少的信息，特别是催化游离蜕皮类固醇磷酸化的酶。本研究的目的是纯化和表征蜕皮甾体激酶（ATP：蜕皮甾体磷酸转移酶）。通过[^3 H]蜕皮激素的磷酸化反应来测定蜕皮甾体激酶。该酶存在于家蚕成熟卵巢的胞浆中，经QSepharose、Chelating Sepharose、Hydroxypatite、Hitrap Q、Mono Q、5 'AMP Sepharose 4 B和Superdex 200 μ g柱层析，纯化倍数约600倍。纯化的酶在pH 7.6时最具活性。这种酶需要ATP和Mg^<2+>同时存在才能表达活性.蜕皮激素激酶的表观动力学参数为Km=3.30 μM（20-羟基蜕皮激素）、3.66 μM（蜕皮激素）、8.22 μM（3-表蜕皮激素）。目前尚不清楚在Schistocerca gregaria中发现的ATP：2-脱氧蜕皮激素22-磷酸转移酶（Kabbouh和Rees 1991）与我们在家蚕中发现的蜕皮激素22-磷酸转移酶（蜕皮激素激酶）是否同源。

项目成果

Makka T.: "A possible role of 20-hydroxyecdysone in embryonic development of the silkworm Bombyx mori"Archives of Insect Biochemistry and Physiology. 51(3). 111-120 (2002)

Makka T.：“20-羟基蜕皮酮在蚕胚胎发育中的可能作用”昆虫生物化学和生理学档案。

Yamada R. and Sonobe H.: "A novel enzyme ecdysteroid-phosphate phosphatase : purification, kinetic characterization, and molecular cloning."J. Biol. Chem.. (in press). (2003)

Yamada R. 和 Sonobe H.：“一种新型脱皮素磷酸酶：纯化、动力学表征和分子克隆。”J.